A number of bacteria are known to produce several secondary metabolites like pigments, toxins, growth promoting compounds and antibiotics (Bushell Mo., 1982, Microbial aspects of the discovery of novel secondary metabolites, Top. Enzyme-Ferment Biotech no 1 6, 32–67). These bacteria have an added advantage in that they are easily replenishable as their generation time is much shorter than other microbes and higher organisms. Besides, their products are eco-friendly and can be used either in their native form or with minor modifications.
The synthetic pigments and dyes have proved to be more harmful than natural products. Some of the synthetic colours are either banned or the permissible concentrations drastically reduced. In the Government of India notification an amendment to the Prevention of Food Adulteration Act in March 1993 banned the use of permitted colours (The Navhind Times dated Aug. 4, 1998) European Union is planning to ban about 300 textile dyes containing chemicals known to increase the risk of cancer. Dyes containing significant levels of 22 of the so-called aromatic amines are known to be carcinogenic (The Navhind Times, dated Oct. 4, 1999). The planned legislation would phase out dyes that are used in textile, leather goods, seat covers, gloves. In short any material having even temporary contact with the skin, public's inclination towards bio pigments and colours is strong.
Pigments are known to be the most important secondary metabolites of organisms for various applications. Even though pigments can be produced by chemical synthesis there has been increasing demand and renewed interest for natural pigments. This is essentially due to the public awareness of the superiority and safety of natural products over the synthetic ones, especially in the food industry.
Reference may be made to the natural colorants like red betalin pigment from plant, Beta vulgaris. (Smith MAL, Dustin I, Leathers R, Zryd Jp 1992. Development of automated vision techniques for immediate analysis and control of betalin producing cultures. Hortiscience 27 (6), 572) or bright colour from the bryozoan animal, Bugula dentata (Matsunaga S, Fusetani N, Hashimoto K, 1986. Bioactive marine metabolites. VIII Isolation of an antimicrobial blue pigment from the bryozoan Bugula dentata Experientia 42(1), 84). However, use of such pigments from higher organisms has got certain limitation. For example the major drawbacks are that higher plants and animals have a longer generation time and the resources are limited. Though tissue culture has evolved as an alternate method to produce the cells for pigment extraction, the techniques are not always simple and have limited application. On the other hand, microbes, especially bacteria, can be exploited more advantageously for reasons mentioned above.
Among the pigments yellow-orange to red pigments, mostly belonging to the carotenoid group has been popular for various applications. For example, B carotene is a valuable metabolite and is in great demand in the food industry and Aquaculture (Michel P J, Dujardin E, Sironval C1995. Growth of Dunalliella bardawil under carotogenic conditions. J. Mar. Biotech no1 2 (2), 101–104). Carotenoids possessing carbonyl groups inhibit lipid peroxidation better than those lacking carbonyl groups (Michel P J, Dujardin E, Sironval C, 1995. Growth of Dunalliella bardawil under carotogenic conditions. J. Mar. Biotechnol 2 (2), 101–104). Despite the enormous economic potential of carotenoids, only a few of the microbes have been exploited commercially. Zeaxanthin, a carotenoid, from a bacterium (Alteromonas sp) is used in the food industry essentially for imparting colour to the product (Japanese patent No. JP-5049497). The same compound from another bacterium Flavobacterium sp (Nelis H J, De Leenheer A P 1991. Microbial sources of carotenoids pigments used in foods and feeds. J of Appl Bacterial 70, 181–191. U.S. Pat. Nos. 841,967, 3,951,742, 3,951,743, 4,026,949) has also been reported. A formulation of zeaxanthin from Flavobacterium multivorans is also claimed to prevent degeneration of macula in the iris of the eye (U.S. Pat. No. 5827652). Astaxanthin, another carotenoid from the yeast, Phaffia rhodozyme (Nelis H J, De Leenheer A P 1991. Microbial sources of carotenoid pigments used in foods and feeds. J of Appl Bacteriol 70, 181–191) has been reported to be useful as growth promoters Another group of pigments like menaquinones extracted from bacteria have also been used for various applications. A process has been claimed for the preparation of menaquinone-containing substance from Bacillus subtilis in food and drink preparations for osteoporosis therapy (JP-11032787). Pigments from Brevibacterium species have been used in the dairy products especially for ripening and flavouring cheese (Reyser E T, Maisnier-Patin S, Gratadoux J J & Richard J 1994. Isolation and identification of cheese smear bacteria inhibitory to Listeria spp. Int. J Food Microbiol. 21, 237–246) and production of menatetrone (JP-63267283; JP-61173792; EP-202613).
Japanese Patent No. 95-05169, titled: “Cosmetic containing fermented Streptomyces sp.” describes “a new cosmetic preparation contains fermentation broth from Streptomyces sp. G172 (FERM P-13630), grown in culture medium at 25–30 deg for 4–7 days. The fermentation broth is extracted and purified to form a clear solution. The cosmetic is useful for whitening skin, or as a sunscreen. In an example G172 was grown at 25 deg for 5 days, centrifuged and maintained at −20 deg overnight to give a precipitate. The precipitate was filtered, and a skin lotion was prepared from the fermentation broth (10 wt. %), glycerol (5%), polyoxyethylene sorbitan monolaurate (1.5%), ethanol (10%), fragrance, antioxidant, antiseptic, pigment and purified water (6 pp)”.
Japanese Patent No.JP07010736; 13.01.95; 128:66319 Coupland, Keith; Packer, Clarie Elizabeth (Croda International PLC; Coupland, Keith; Packer, Claire Elizabeth, UK) titled: “Sunscreen compositions comprising stearidonic acid and derivatives in combination with a UV blocking and/or absorbing material”, describes “A sunscreen composition comprising a stearidonic acid, or a physiol. deriv. thereof, in combination with a UV blocking and/or UV absorbing material, is claimed. Also stearidonic acid may be used to treat inflammation caused by exposure to UV radiation, by exposure to sunlight or by burns. Thus, 10 kg of the seeds of Echium plantagineum were crushed and the oil was extracted with 15 L of petroleum ether. The petroleum ether extract was evaporated to yield 1741 g of golden yellow oil. The oil was converted to the corresponding fatty acid Me esters and used in sunscreens. A sunscreen oil was prepared containing Bu methoxydibenzoyl methane 2.0, octyl methoxy-cinnamate 7.5, benzophenone-3 4 5, PPG-2 myristyl ether propionate 10.0, above oil 2.0–10.0, perfumes, preservatives, and caprylic/capric triglycerides q.s. 100%.
Sophie Maisnier-patin and J. Richard, Station de Recherches Laitieres, Institut National de la Recherche Agronomique, Jouy-en-Joses, France, (Applied and Environmental Microbiology, May 1995, p. 1847–1852); titled: Activity and Purification of Linenscin OC2, an Antibacterial Substance Produced by Brevibacterium linens OC2, an Orange Cheese Coryneform Bacterium, describes: An orange cheese coryneform bacterium isolated from the surface of Gruyere of Comte and identified as Brevibacterium linens produces an antimicrobial substance designated linenscin OC2. This compound inhibits gram-positive food-borne pathogens including Staphylococcus aureus and Listeria monocytogenes but is not active against gram-negative bacteria”.
Japanese Patent No.99-06310; titled: “A process for preparation of menaquinone-7 containing substance and food and drink preparations” describes: “A process is claimed for the preparation of menaquinone-7 (1) for use in food and drink preparations. (1) is produced by culturing Bacillus subtilis MR-141 (FERM P-14692), S-2 (IFO 14898, FERM BP-2528), ATCC 162 (FERM P-11052), F-2-01 (FERM P-9082) or their mutants in a liquid nutrient media at 30–40 deg, pH 6–8 for 2–4 days. The cells are removed by filtration and centrifuged. Both the cell pellet and the supernatant are dehydrated (e.g. by spray drying, lyophilization and drying in vacuo) in the absence of an organic solvent. The product is used for the prevention and treatment of osteoporosis (no clinical data disclosed) and is included in dried foods at 0.1–10% and drink preparations at 0.1–5%”.
Most of the commercially available UV-blocking compounds in skin cream (sunscreen) are synthetic and the search for natural compounds with equal or greater efficiency is becoming more significant because of the consumer's preference for natural products.
The UV-absorbing properties of either the organisms or the extract have been extensively studied in higher plants, corals, cyanobacteria and other phytoplankton. Reference may be made to an UV absorbing (310 nm) compound that has been characterised from stem, bark and roots of mangrove plant, Heritiera littoralis (Bandaranayake WM 1994 phyto-chemical constituents and pigments in mangrove species and mangal associates of Northern Australia. Aust Inst Mar Sci Rep Townsville, Old Australia AIMS 19, 28 pp). The hyperoxic tissues of coral reefs also produce UV absorbing mycosporine like compounds (Dunlap WC Shick JM 1998. Ultraviolet radiation absorbing mycosporine-like amino acids in coral reef organisms: A biochemical and environmental perspective. J. Phycol. 34(3), 418–430). The induction and protective role of the UV-absorbing compounds such as mycosporine-like amino acids (MAAs) have been noted even in Florideophyceae (Franklin La., Yakovleva. I, Karsten U, Luenig, K 1999. Synthesis of mycoporine-like aminoacids in Chondrus crispus(Florideophyceae) and the consequences for sensitivity to ultraviolet B radiation. J. Phycol. 35, 682–693) Certain algae like Dunaliella are also known to possess intense carotenoid pigments to protect themselves against intense solar radiation. Some algae have other type of UV-absorbing (sunscreen) pigments like scytonemin (Proteau, P. J., Gerwick, W. H., Garcia-Pichel F. Castenholz 1993. The structure of scytonemin an ultraviolet sunscreen pigment from the sheath of cyanobacteria. Experientia 49, 825–829). These UV absorbing compounds are also known to be produced under photoinductive conditions and are dependant on temporal factors (Hannach G, Sigleo AC 1998. Photoinduction of UV absorbing compounds in six species of marine phytoplankton. Mar. Ecol. Prog Ser 174; 207–222). Though a number of papers have been published on the ultraviolet-absorbing/screening substances in cyanobacteria, phytoplankton and macroalgae and their role in mitigating ultraviolet toxicity (Sinha R P, Klisch M, Groeniger A, Haeder D P. 1998. Ultraviolet absorbing/screening substances in cyanobacteria, phytoplankton and macroalgae. J Photochem Photobiol B 47 J (2–3), 83–94) there have been only few publications on this aspect of bacteria (Arai T. Nishijima M, Adachi K Sano H 1992. Isolation and structure of a UV absorbing substance from the marine bacterium Micrococcus sp AK 334. Marine Biotech Inst Rep. Pp88–94 Japan). Sunscreen compositions comprising natural products of marine hydroid and derivatives thereof have been patented as useful sunscreening agents (lindquist, N.1. 1998 U.S. Pat. No. 5,705,146). A sunscreen/radioprotective compound has also been patented from fungus Aspergillus versicolor FK17 95-03294 (JP-06329576). Though bacteria possess a number of pigments that are supposedly photo protective only a few have been used for extracting UV A and B absorbing components. A fat-soluble UV absorbing compound F-1547 from Paracoccus sp has been patented (JP-11269175). A process for producing UV absorbing mycosporine-like aminoacids (MAA) from Micrococcus sp has also been developed (JP-06062878-A).
Antibacterial activity is widespread in microorganisms especially in those belonging to streptomycete group and many compounds from this group have been discovered and the processes and products have been patented. Ditrisaburicins A, B, and C from Streptomyces cyaneus are active against Gram-positive bacteria (Microb. Chem. Res. Found. EP-110155) For example CV 1 which is an antibacterial compound from Streptomyces sp is also used for sterilising instruments in hospitals (JP-J62000286). AAD-609 antibiotics produced by the fungus Kibdelosporangium aridum ATCC 3922 are inhibitory to Gram-positive bacteria (EP-218416). Narrow spectrum bactriocins from bacteria that are active against closely related species is common. Bacteriocins have wide-ranging applications in the food industry as preservatives (Eckner, K. F. 1992. Bacteriocins and Food Application. Dairy Food and Environ. Sanitation 12 (40), 204–209). Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It is bacteriolytic to Listeria innocua. (Boucabeille C. Menginlecreulx D. Henckes G. Simonet J M, Vanheijenoort J. 1997. Antibacterial and haemolytic activities of linocin OC2 a hydrophobic substance produced by Brevibacterium linens OC2. FEMS Microbiology Letters 153 (2), 295–301). The compound inhibits Gram-positive food-borne pathogen including Staphylococcus aureus and Listeria monocyclogenes but is not active against Gram-negative bacteria. A process for the production of antibacterial compound difficidin and its derivative is described where oxydifficidin is produced in large amounts compared with the other derivatives (U.S. Pat. No. 4,545,991; Zimmerman Sb, Schwartz C D, Monaghan R L, Pelak B A, Gillifillan E C 1987. Difficidin and Oxydifficidin; a novel broad-spectrum antibacterial antibiotics produced by Bacillus subtilis preparation and characterisation. J. Antibiot, 40 (12), 1677–1681.) These compounds have been shown to be active against Klebsiella pneumonie. Antibacterial compounds from bacteria, which are active against both Gram-positive and Gram-negative groups, are uncommon.
Some of the pigments in bacteria are also associated with antimicrobial activity. The use of antibiotic pigment violcacein isolated from Janthinobacterium lividum (J P-10113169) or Chromobacterium violaceum and Janthinobacterium (JP-O6253864) has been patented. Pigments with antimicrobial properties have been isolated even from photosynthetic bacteria, Chromatium purpuratum (Burgess J G, Miyashita H, Sudo H, Matsunaga T 1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704:localisation of the activity to the chromatophores FEMS Microbiol lett. 84 (3), 301–06). Cultures of aerobic marine bacterium Alteromonas rubra produce red pigments with pH indicator properties and a compound showing antibiotic activity against a variety of bacteria (Laatsch H and Thomson RH 1983. A revised structure for cycloprodigiosin. Tetrahedron Lett. 24 (26), 2701–04).
Though there have been reports on the occurrence of pigments alone or pigments with antibacterial activity in some of the microbes and sun screen compounds from yet others, there is no report available on the presence of all three in a single extract of a single bacterial species.